Isovaleryl Sucrose Esters from Atractylodes japonica and Their Cytotoxic Activity

Cancer represents one of the most significant health challenges currently facing humanity, and plant-derived antitumour drugs represent a prominent class of anticancer medications in clinical practice. Isovaleryl sucrose esters, which are natural constituents, have been identified as having potential antitumour effects. However, the mechanism of action remains unclear. In this study, 12 isovaleryl sucrose ester components, including five new (1–5) and seven known compounds (6–12), were isolated from the roots of Atractylodes japonica. The structures of the compounds were elucidated using 1D and 2D-NMR spectroscopy, complemented by HR-ESI-MS mass spectrometry. The cytotoxic activities of all the compounds against human colon cancer cells (HCT-116) and human lung adenocarcinoma cells (A549) were also evaluated using the CCK8 assay. The results demonstrated that compounds 2, 4, and 6 were moderately inhibitory to HCT-116 cells, with IC50 values of 7.49 ± 0.48, 9.03 ± 0.21, and 13.49 ± 1.45 μM, respectively. Compounds 1 and 6 were moderately inhibitory to A549, with IC50 values of 8.36 ± 0.77 and 7.10 ± 0.52 μM, respectively. Molecular docking revealed that compounds 1–9 exhibited a stronger affinity for FGFR3 and BRAF, with binding energies below −7 kcal/mol. Compound 2 exhibited the lowest binding energy of −10.63 kcal/mol to FGFR3. We screened the compounds with lower binding energies, and the protein-ligand complexes already obtained after molecular docking were subjected to exhaustive molecular dynamics simulation experiments, which simulated the dynamic behaviour of the molecules in close proximity to the actual biological environment, thus providing a deeper understanding of their functions and interaction mechanisms. The present study provides a reference for the development and use of iso-valeryl sucrose esters in the antitumour field.


Introduction
Cancer is one of the most significant health challenges currently facing humanity [1], and plant-derived antitumour drugs represent a prominent class of anticancer medications in clinical practice [2].Examples of such drugs include the topoisomerase inhibitor camptothecin [3], the antimitotic tumour drug paclitaxel [4], and the tumour differentiation and apoptosis-inducing elemene [5].Consequently, the discovery and isolation of targeted antitumour drugs from traditional Chinese medicine with the potential for mass production, high efficiency, and low toxicity has been a subject of intense research interest for numerous scientists.
Atractylodes japonica, a member of the genus Atractylodes DC belonging to the family Asteraceae, is a native medicinal herb in northeast China and is mainly distributed in the Jilin, Heilongjiang, and northeast Liaoning Provinces [6].This herb is widely used in northeast China for the treatment of ailments such as spleen pain, abdominal pain, diarrhoea, lethargy, and weakness [7].According to the existing literature [8], A. japonica contains a type of sucrose ester with only isovaleryl as a substituent, which is rare in natural products.Sucrose esters are non-ionic surfactants often produced in the laboratory from sucrose and fatty acids by esterification.They exhibit a range of biological activities, including antibacterial, anti-inflammatory, antitumour, and insecticidal activities [9,10].Sucrose esters with only isovaleryl as a substituent are relatively rare among natural products, with only 25 compounds reported to date.Of these, 21 are from the Asteraceae family, including seven from A. lancea [11,12], four from A. japonica [8] and eight from A. yunnanensis.Among these, ainsloside B showed remarkable cytotoxicity against A549 lung cancer cells with an IC 50 value of 3.3 µM [13].In addition, two compounds have been identified in Chrysanthemum maculatum [14].Only four isovaleryl sucrose esters have been reported from other families.Three of these are from the roots of Brassica rapa ssp.campestris of the Brassicaceae family [15], while the fourth is from Euphorbiae Lathyridis Semen of the Euphorbiaceae family [16], which has been shown to inhibit melanogenesis.It can be observed that these ingredients exhibit cytotoxic activity against a range of tumour cells.However, there is a paucity of literature on the mechanism of action of these compounds.The BRAF and FGFR genes are aberrantly expressed in a variety of cancers, as evidenced by studies [17,18].According to literature reports, aberrant expression of BRAF is present in 4.7-22.6% of patients with colorectal cancer [19,20]; Aberrant expression of FGFR3 can lead to the development and progression of lung cancer, particularly non-small cell lung cancer [21,22].Moreover, BRAF inhibitors [23,24] and FGFR inhibitors [25][26][27] are currently the subject of intense research, with some of these inhibitors already being applied in clinical practice.Therefore, these two proteins were the focus of this study.
Therefore, the aim of this study was to isolate and characterise isovaleryl sucrose esters from Atractylodes japonica and to investigate their cytotoxic activity.To explore the mechanism of action of all compounds, we subjected all the compounds to molecular docking and molecular dynamics simulations with FGFR3 and BRAF proteins, respectively.
In this study, 12 isovaleryl sucrose esters were isolated from A. japonica (Figure 1), including five new isovaleryl sucrose esters (1)(2)(3)(4)(5) and seven known isovaleryl sucrose esters (6)(7)(8)(9)(10)(11)(12).The cytotoxic activities of all the compounds against colon cancer cells HCT-116 and lung adenocarcinoma cells A549 were evaluated using the Cell Counting Kit-8 (CCK-8) assay.The results showed that compounds 2, 4, and 6 were moderately inhibitory to HCT-116 cells, with IC 50 values of 7.49 ± 0.48, 9.03 ± 0.21, and 13.49 ± 1.45 µM, respectively.Compounds 1 and 6 were moderately inhibitory to A549, with IC 50 values of 8.36 ± 0.77 and 7.10 ± 0.52 µM, respectively.The results show that compounds 1-10 have lower binding energies to these two proteins, all below −7 kcal/mol.We carried out molecular dynamics simulation experiments with compounds 2, 4, 6-8, which have lower binding energies, and the results show that the binding of these compounds to the proteins is relatively stable.The results suggest that such compounds may exert cytotoxic activity against HCT-116 and A549 cells by acting on FGFR3 and BRAF proteins.Because these compounds are easy to synthesise in the laboratory, they have some development potential.

Stability Analysis of Small Molecule-Protein Receptor Complexes
Root-mean-square deviation (RMSD) [33] curves are of significant importance in molecular dynamics simulations, serving as a crucial indicator for the assessment of structural stability in protein-ligand complexes.
As illustrated in Figure 6A, the RMSD curve of compound 2-BRAF (blue line) remains relatively stable at approximately 0.45 nm for a duration of 15-100 ns.The RMSD curve of compound 4-BRAF (black line) is stable around 0.45-0.50nm for 35-100 ns, and the RMSD curve of compound 6-BRAF (red line) indicates that the compound is relatively stable at 0.45-0.50nm for 35-100 ns, which suggests that compounds 2, 4, and 6 can form

Stability Analysis of Small Molecule-Protein Receptor Complexes
Root-mean-square deviation (RMSD) [33] curves are of significant importance in molecular dynamics simulations, serving as a crucial indicator for the assessment of structural stability in protein-ligand complexes.
As illustrated in Figure 6A, the RMSD curve of compound 2-BRAF (blue line) remains relatively stable at approximately 0.45 nm for a duration of 15-100 ns.The RMSD curve of compound 4-BRAF (black line) is stable around 0.45-0.50nm for 35-100 ns, and the RMSD curve of compound 6-BRAF (red line) indicates that the compound is relatively stable at 0.45-0.50nm for 35-100 ns, which suggests that compounds 2, 4, and 6 can form a stable complex system with BRAF.As shown in Figure 7A, the RMSD curve of compound 2-FGFR3 (blue line) is stable around 0.33 nm for 30-100 ns.The RMSD curve of compound 7-FGFR3 (red line) is relatively stable around 0.36-0.40nm for 60-100 ns.The RMSD curve of compound 8-FGFR3 (black line) is usually stable around 0.28-0.32nm for 10-100 ns, indicating that compounds 2, 7, and 8 can form stable complex systems with FGFR3.The RMSD provides a statistical measure to assess the degree of difference between the predicted structure and the experimentally determined protein structure.It can be seen that these RMSD curves gradually levelled off during the simulation, indicating that the structures of these complexes fluctuated less during the simulation, i.e., the complexes were more stable.
Molecules 2024, 29, x FOR PEER REVIEW a stable complex system with BRAF.As shown in Figure 7A, the RMSD curve o pound 2-FGFR3 (blue line) is stable around 0.33 nm for 30-100 ns.The RMSD c compound 7-FGFR3 (red line) is relatively stable around 0.36-0.40nm for 60-100 RMSD curve of compound 8-FGFR3 (black line) is usually stable around 0.28-0.3210-100 ns, indicating that compounds 2, 7, and 8 can form stable complex system FGFR3.The RMSD provides a statistical measure to assess the degree of differe tween the predicted structure and the experimentally determined protein structure be seen that these RMSD curves gradually levelled off during the simulation, ind that the structures of these complexes fluctuated less during the simulation, i.e., th plexes were more stable.Root-mean-square fluctuation (RMSF) curves are key indicators of the dynamic behaviour of individual amino acid residues in proteins during molecular dynamics simulations [34].As illustrated in Figure 6B, BRAF exhibits greater residue flexibility at residues 470-485, 490-495, 530-555, 590-660, 530-555, 590-610, and 620-635.As illustrated in Figure 7B, FGFR3 displays greater residue flexibility at residues.The following ranges were identified as exhibiting greater residue flexibility: 470-480, 490-500, 510-520, 540-550, 570-590, and 640-660.The analysis of the RMSF curves enables a more comprehensive understanding of the dynamic behaviour of the receptor protein during molecular dynamics simulations, particularly in relation to residue fluctuations upon binding of small molecules to the protein.These highly fluctuating residues may play a significant role in the function and activity of proteins, providing a valuable reference for subsequent biochemical studies and drug design.The radius of gyration (Rg) is a crucial parameter for evaluating the structural compactness and stability of proteins or other biomolecules [35].Figure 6C demonstrates that the Rg value of BRAF remains stable over 10-100 ns, with the lowest average Rg value ranging from 2.68 to 2.80 nm.The Rg values of compound 2 (blue line) are shown to be stable over 35-100 ns, with the lowest average Rg values ranging from 2.68 to 2.75 nm.The Rg value of compound 7 (red line) remained stable over 0-100 ns, with the lowest average Rg value ranging from 2.67 to 2.75 nm.The Rg value of compound 8 (black line) remained stable over 35-100 ns, with the lowest average Rg value ranging between 2.72 and 2.80 nm. Figure 7C illustrates that the Rg value of FGFR3 remains consistent within the 30-100 ns timeframe, with the lowest average Rg value ranging from 1.98 to 2.08 nm.The Rg value of compound 2 (blue line) remained stable over 70-100 ns, with the lowest average Rg value ranging from 1.97 to 2.07 nm.The Rg value of compound 7 (red line) was shown to be stable over 30-100 ns, with the lowest average Rg value ranging from 1.97 to 2.06 nm.The Rg value of compound 8 (black line) is shown to be stable over 60-100 ns, with the lowest average Rg value ranging from 2.00 to 2.08 nm.This indicates that the protein complex retained a relatively stable spatial conformation throughout the kinetic simulations, demonstrating minimal structural expansion or contraction.This result is crucial for elucidating the stability and interaction mechanisms of the complex.The smooth radius of gyration curves suggest that the complexes were able to maintain their structural integrity during the simulations.
In order to gain a more detailed understanding of the conformational changes of the two proteins during the interaction, we conducted an exhaustive analysis of the 100-nanosecond molecular dynamics simulation data, with a particular focus on the hydrogen The radius of gyration (Rg) is a crucial parameter for evaluating the structural compactness and stability of proteins or other biomolecules [35].Figure 6C demonstrates that the Rg value of BRAF remains stable over 10-100 ns, with the lowest average Rg value ranging from 2.68 to 2.80 nm.The Rg values of compound 2 (blue line) are shown to be stable over 35-100 ns, with the lowest average Rg values ranging from 2.68 to 2.75 nm.The Rg value of compound 7 (red line) remained stable over 0-100 ns, with the lowest average Rg value ranging from 2.67 to 2.75 nm.The Rg value of compound 8 (black line) remained stable over 35-100 ns, with the lowest average Rg value ranging between 2.72 and 2.80 nm. Figure 7C illustrates that the Rg value of FGFR3 remains consistent within the 30-100 ns timeframe, with the lowest average Rg value ranging from 1.98 to 2.08 nm.The Rg value of compound 2 (blue line) remained stable over 70-100 ns, with the lowest average Rg value ranging from 1.97 to 2.07 nm.The Rg value of compound 7 (red line) was shown to be stable over 30-100 ns, with the lowest average Rg value ranging from 1.97 to 2.06 nm.The Rg value of compound 8 (black line) is shown to be stable over 60-100 ns, with the lowest average Rg value ranging from 2.00 to 2.08 nm.This indicates that the protein complex retained a relatively stable spatial conformation throughout the kinetic simulations, demonstrating minimal structural expansion or contraction.This result is crucial for elucidating the stability and interaction mechanisms of the complex.The smooth radius of gyration curves suggest that the complexes were able to maintain their structural integrity during the simulations.
In order to gain a more detailed understanding of the conformational changes of the two proteins during the interaction, we conducted an exhaustive analysis of the 100-nanosecond molecular dynamics simulation data, with a particular focus on the hydro-gen bonding formed between the two proteins.As illustrated in Figure 7D.The number of hydrogen bonds exhibited fluctuations throughout the simulation.At the initial stage (0 to 25 ns), the number of hydrogen bonds was relatively low, with an average of between 1 and 4. As the simulation progresses, the number of hydrogen bonds gradually increases, reaching a peak value between 7 and 10 between 20 and 60 ns.This phenomenon indicates that the protein-compound interaction is enhanced during this time period, resulting in the formation of additional hydrogen bonds.This result indicates the existence of a stable yet dynamic hydrogen bonding network between the two proteins.Despite the fluctuations in the number of hydrogen bonds, the overall number remained at a relatively high level, indicating a stronger and more stable interaction between the proteins.This dynamic equilibrium is of great importance in maintaining the overall structure of the protein complex and ensuring its proper function.

Gibbs Free Energy Analysis
In this study, advanced computational methods and visualisation tools have been employed in order to conduct a comprehensive investigation into the stability and interaction mechanisms of small molecule-protein receptor docking complexes [36,37].This was based on the RMSD (root-mean-square deviation) and Rg (radius of gyration) values of the complexes.The Gibbs free energy was calculated accurately.As illustrated in Figure 8, compounds 2, 4, and 6 exhibit a steady state with the BRAF protein at PC1 0.23-0.50and PC2 2.64-2.77.The region with a blue hue in the figure prominently labels the steady-state conformations of the complexes, which are clustered at lower energies within the minimum free energy region.As illustrated in Figure 9, compounds 2, 7, and 8 exhibit a stable interaction with the FGFR3 protein at PC1 0.22-0.50and PC2 1.94-2.04.The smoothness and depth of this region reflect the strength of its complex stability.This further corroborates the excellent stability of small-molecule protein receptor complexes, which is crucial for drug design and biomedical research.The complexes are able to maintain their structural and functional integrity under physiological conditions, thus exerting their biological activities more efficiently.

MM/GBSA and Binding Free Energy Analysis
In this study, to further understand the degree of binding stability of small molecules to target protein receptor molecules, the MM/GBSA method was used to calculate the binding free energy and the free energy contributions of the amino acid residues [38].The binding components in the free energy of binding small molecules to protein receptors are summarised in (Tables 4 and 5).The free energies of binding of Compound 2-FGFR3, Compound 7-FGFR3, and Compound 8-FGFR3 were −44.43 ± 6.00, −37.23

MM/GBSA and Binding Free Energy Analysis
In this study, to further understand the degree of binding stability of small molecules to target protein receptor molecules, the MM/GBSA method was used to calculate the binding free energy and the free energy contributions of the amino acid residues [38].The binding components in the free energy of binding small molecules to protein receptors are summarised in (Tables 4 and 5).  5.

Discussion
In the present study, 12 sucrose esters were isolated and purified from Atractylodes japonica, including five new compounds and seven known compounds.This study greatly enhanced the presence of this class of components in natural products.In addition, the results of the cytotoxic activity demonstrated that these compounds exhibited activity against colon cancer and lung cancer cells.The superior cell toxicity and ability to bind to BRAF and FGFR3 of compounds 1-7 in comparison to compounds 8-12 suggest that the activity of sucrose esters with three isovaleryl substitutions is superior to that of sucrose esters with five isovaleryl substitutions.This provided a theoretical basis for the subsequent structural modification of these compounds.Additionally, the results of molecular dynamics simulations indicated that the binding energies and thermodynamic stability between the small molecules and protein receptors were enhanced.Moreover, the laboratory synthesis of this class of compounds is relatively straightforward, rendering it highly valuable for intensive research and exploitation.

Plant Materials
The rhizomes of A. japonica were collected in October 2021 from the Daxinganling region in Heilongjiang Province, China.Professor Wang Zhenyue authenticated these rhizomes as Atractylodes japonica.A specimen has been archived at the School of Pharmacy, Heilongjiang University of Chinese Medicine, Harbin, China, with the reference number 20211007.

Cytotoxicity Assays
The human colorectal carcinoma (HCT-116) and human non-small cell lung carcinoma (A549) cells were cultured in high-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% foetal bovine serum (FBS) and 1% penicillin-streptomycin.In brief, the two types of cells were seeded in flat-bottomed 96-well microplates at a density of 4000 cells per well, with a total volume of 0.2 mL per well.Following a 24-h incubation period, the cells were exposed to varying concentrations of the compounds (1-12) for a further 48 h.The concentration range of the compounds was between 0 and 100 µM.After 24 h of incubation, the existing medium was replaced with 100 µL of fresh medium containing 0.5 mg/mL of CCK-8 reagent.Subsequently, the cells were incubated for a further two hours under the same conditions.Finally, the absorbance was measured at 490 nm in order to calculate the IC 50 value, as previously reported in the literature [39].The IC 50 value was deemed to be greater than 50 µM, indicating that the compound was inactive or ineffective.Doxorubicin was used as a positive control.

Molecular Docking Analysis
The crystal structures of FGFR3 (PDB ID: 6LVM) and BRAF (PDB ID: 8C7X) were obtained from UniProt (https://www.uniprot.org,accessed on 2 April 2024).The receptor was prepared using PyMOL 2.5.5 and deposited in .pdbformat.The 3D structures of the ligands were energy optimised using ChemDraw 3D 14.0 and deposited in mol2 format.The ligands and receptors for molecular docking analysis were prepared using AutoDock Vina 1.2.2 [33] The results of the docking calculations were analysed using PyMOL 2.5.5 [39][40][41].2D visualisation analysis was performed using ligplus 2.2.8 software [42].

Molecular Dynamics Simulation Analysis
Molecular dynamics simulations were conducted to evaluate the binding stability, conformation, and interaction pattern of compounds 2, 7, 8 with the receptor FGFR3 (PDBID: 6LVM) and compounds 2, 4, and 6 with the receptor BRAF (PDBID: 8C7X), respectively.The software employed was Gromacs 2022.3 [43,44], with the simulation conditions maintained at a static temperature of 300 K.The force field was Amber99sb-ildn, the solvent was a water molecule (Tip3p water model), and the equilibrium duration was 100 ps.Finally, molecular dynamics simulations were carried out for 100 ns.Finally, we analysed each set of molecular dynamics simulation trajectories for the root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), number of hydrogen bonds, and Gibbs free energy diagrams.These analyses were conducted in accordance with the methodologies outlined in references [45][46][47][48].

Conclusions
In the present study, 12 sucrose esters were isolated and purified from Atractylodes japonica, including five new compounds and seven known compounds.The results of cytotoxicity assays show that compounds 2, 4, and 6 were moderately inhibitory to HCT-116 cells, with IC 50 values of 7.49 ± 0.48, 9.03 ± 0.21, and 13.49 ± 1.45 µM, respectively.Compounds 1 and 6 were moderately inhibitory to A549, with IC 50 values of 8.36 ± 0.77

Figure 5 .
Figure 5. Visualisation results of molecular docking of compounds to proteins: (A) is compound 2 with BRAF (binding energy −9.95 kcal/mol), (B) is compound 4 with BRAF (binding energy −10.42 kcal/mol), (C) is compound 6 with BRAF (binding energy −9.27 kcal/mol), (D) is compound 2 with FGFR3 (binding energy −10.63 kcal/mol), (E) is compound 7 with FGFR3 (binding energy −10.45 kcal/mol), and (F) is compound 8 with FGFR3 (binding energy −10.48 kcal/mol).(In the three-dimensional diagram, green represents small molecule ligands, blue represents receptor proteins, and purple represents amino acid residues interacting with the ligand.In the two-dimensional diagram, purple represents ligands and red represents amino acid residues interacting with the ligand).

Figure 6 .
Figure 6.Molecular dynamics simulation results: graph (A) RMSD of compounds 2, 4, and BRAF.(B) RMSF of compounds 2, 4, and 6 with BRAF.(C) Rg of compounds 2, 4, and 6 wit (D) H bond number of compounds 2, 4, and 6 with BRAF.Root-mean-square fluctuation (RMSF) curves are key indicators of the dyna haviour of individual amino acid residues in proteins during molecular dynamic lations[34].As illustrated in Figure6B, BRAF exhibits greater residue flexibility dues 470-485, 490-495, 530-555, 590-660, 530-555, 590-610, and 620-635.As illustr Figure7B, FGFR3 displays greater residue flexibility at residues.The following were identified as exhibiting greater residue flexibility: 470-480, 490-500, 510-52 550, 570-590, and 640-660.The analysis of the RMSF curves enables a more comp sive understanding of the dynamic behaviour of the receptor protein during mo dynamics simulations, particularly in relation to residue fluctuations upon bin small molecules to the protein.These highly fluctuating residues may play a sig role in the function and activity of proteins, providing a valuable reference for subs biochemical studies and drug design.